In the John Wayne Cancer Institute, active immunotherapy of melanoma by allogeneic tumor cell (PMCV) vaccination has shown enhanced survival of patients who developed an immune response but only a fraction demonstrates the regression of established metastases. Alternatively, the therapeutic efficacy may be improved if isolated tumorimmune T cells are transferred to the tumor-bearing host. We have identified that lymph nodes draining tumor vaccines contain T lymphocytes specifically sensitized to the tumor, but those cells require in vitro activation to differentiate into functional cells. We have designed a clinical protocol to treat patients with metastatic melanoma with such T cells. Although responses were observed in some patients, the majority did not seem to benefit. A theoretically attractive improvement for both active and adoptive immunotherapy will rely on targeted vaccine designs. To induce antigen (Ag)- specific immune responses, host Ag presenting cells (APC) play a pivotal role. Among various APC, dendritic cells (DC) seem to be most potent for eliciting T cell responses. The use of DC for stimulating antitumor immunity has been achieved by pulsing DC with peptides, protein, tumor lysates or RNA derived from neoplastic cells. A new approach is the generation of DC-tumor fusion hybrids. Because of technical difficulties, attempts have been made to fuse cells by exposing them to electric fields. Recently, a clinical trial treating patients with metastatic renal cancer with electrofusion of allogeneic DC and autologous tumor cells demonstrated a high response rate (7/17). Fused hybrids should have the ability to elicit both MHC class I- and II-restricted responses by processing and presenting both known and undefined Ag. With this approach, it may be the technique rather than conceptual aspects that limit its transfer to clinic. In animal studies, we have demonstrated that a high fusion efficiency (~35%) by electrofusion. Using PMCV, preliminary results showed at least a 10% fusion rate. In this project, we propose to carry out Phase I/Ii clinical trials and address important questions regarding the biological and immunological functions of autologous DC-allogeneic tumor hybrids generated by electrofusions. The specific aims are: 1) To optimize electrofusion of autologous DC and allogeneic melanoma PMCV cells; 2) To characterize the functions of fused cells for Ag processing and presentation as well as MHC restriction; 3) To conduct Phase I/II clinical trials of immunotherapy of melanoma; 4) To analyze humoral and cellular immune responses of patients undergoing immunotherapy.